Problems to find receptor ligand mimetics. Modulation of biological functions through agonistic or antagonistic receptor ligands typically aims at either blocking or moderating overtly active biochemical pathways or activating or restoring pathways that are believed to relate to or are causative to a certain disease.
Different to endogenous or xenobiotic proteins and antibodies that exhibit very often highly specific biological activities, small molecules are much less specific. Due to their limited size, small molecules bind to a number of different targets, which may lead to undesired side effects and makes their development as therapeutically useful agents a challenge. For example, careful preclinical and clinical studies are needed to establish a therapeutically useful dose window, to discover off-target effects and their implications in drug development. Particularly the discovery of selective small molecules that are functional mimetics of protein receptor ligands presents a challenge to drug discovery, as the interaction sites and their area between the receptor and its natural ligand are exceeding by far the interaction area provided by small molecules (P. Chene, Chem. Med. Chem. 2006, 1: 400-411). Prototype molecules that have been found in primary biological screens have often the potential to also interact with other receptors and being non-specific on a cellular level.
Moreover, while it has been possible to find small molecules that are antagonists of a given receptor/ligand interaction, it is considered more challenging to identify agonists. Therefore, there is a clear need for a method that may distinguish selective and functional small molecule receptor ligand mimetics from molecules that are “promiscuous” by binding to multiple targets in an undesired way.
Problems to find useful combination treatments. While significant advancements have been made in many diseases and especially in the therapy of tumors there is often still a limited benefit provided by single agents as monotherapies. This is not surprising, given that molecular pathways that are responsible for diseases are often redundant and variable between individual patients or between cell subclones within the same patient. It is therefore often unlikely that a treatment focusing on a single target would offer durable disease control or therapy in most patients (J. E. Dancey and H. X. Chen, Nat. Rev. Drug Discov. 2006, 5:649-659). The use of drug combinations offers a well-established therapy principle, especially in cancer therapy, to provide a better therapy and benefit to patients. With few exceptions, useful drug treatments for cancer use a combination of agents of known activity and minimally overlapping spectra of toxicity, at their optimal doses and according to schedules that are compatible with normal cell recovery. Only few of those chemotherapeutic combinations have been critically evaluated pre-clinically and still fewer of these combinations are synergistic—providing greater benefit in their combination than by the additive effects of their individual activities. Many current combination treatments have been first tried in clinical studies—using a trial-and-error approach with human patients.
This largely empirical approach in clinical development towards combination therapies has been justified by the lack of means of identifying, which tumors might be sensitive to a combination of individual agents: There is a considerable lack of correlation between the outcomes of laboratory in vitro and in vivo experiments and clinical human studies because of the inherent limitations of in vitro and in vivo disease models. In addition, permanent cancer cell lines as for example provided by organizations like the ATCC or used by the National Cancer Institute show considerable alterations in biological properties and chemosensitivity patterns when compared with the original tumors from which they derive. Two studies have shown the limited correlation between in vitro testing in the 60-cell-line panel in the National Cancer Institute, in vivo xenografts and clinical efficacy of cytotoxic agents (J. I. Johnson, Br. J. Cancer 2001, 84: 1424-1431; T. Voskoglou-Nomikos et al., Clin. Cancer Res. 2003, 9: 4227-4239).
Second, methods are also needed to provide information on optimal treatment sequences of the combination. For example, laboratory studies have revealed that the EGFR inhibitor gefitinib is more effective in vitro in combination with standard cytotoxic agents given as a highdose ‘pulse’ prior to paclitaxel when compared with continuous, concurrent administration (D. B. Solit, Clin. Cancer Res. 2005, 11: 1983-1989).
Therefore, methods to rationally design and to experimentally validate synergistic drug combinations are urgently needed as they are expected to provide larger and durable therapeutic benefit for cancer patients.
Apoptosis is mediated through membrane bound receptors. Programmed cell death, termed apoptosis mediates the maintenance of tissue homeostasis, regulating removal of cells from the skin and gastrointestinal tract and remodeling of bone in response to environmental triggers. Apoptosis also prevents diseases by inhibiting systemic viral infection, deleting self-reactive T cells and B cells to prevent autoimmunity, and ablating cells that acquire potentially oncogenic characteristics. Dysregulation of apoptosis plays a role in many disease processes. Inappropriate activation of apoptosis is associated with neurodegenerative disorders such as Parkinson's and Alzheimer's disease, or myocardial damage seen after reperfusion of cardiac tissue following an infarct. Furthermore, if foreign epitopes are detected on the cell surface, apoptosis can be selectively induced by natural killer (NK) cells or cytotoxic T lymphocytes (CTL) of the immune system. Resistance to apoptosis or a higher threshold at which cells undergo apoptosis is associated with mutations in many genes, such as the p53 tumor suppressor gene. Hyperproliferating cells, which have defects in apoptotic pathways, may demonstrate a survival advantage, leading to further malignant progression and ultimately cancer.
The intrinsic (or mitochondrial) and the extrinsic apoptosis (or receptor mediated) pathway are two mechanisms through which apoptosis can occur.
In the intrinsic pathway, the functional consequence of proapoptotic signaling is the perturbation of the mitochondrial membrane and the release of cytochrome c into the cytoplasm. Here, cytochrome c forms a complex with apoptotic protease activating factor 1 (APAF1) and the inactive form of caspase-9, termed the apoptosome. This complex hydrolyzes adenosine triphosphate to cleave and activate caspase-9. This initiator caspase proceeds to cleave and activate the executioner molecules, caspase-3, caspase-6, and caspase-7. Release of cytochrome c from mitochondria is an early event of apoptosis that precedes caspase and endonuclease activation. The release of cytochrome c also leads to the formation of reactive oxygen species (ROS).
Apoptosis induced by the extrinsic apoptotic pathway is mediated through the activation of cell surface death receptors. After ligand mediated activation of a death receptor, the conserved intracellular death domain attracts the intracellular adapter molecule, Fas-associated death domain (FADD). The adapter molecule recruits caspase-8 and caspase-10 to the death receptor, forming the death-inducing signal complex (DISC), where they are cleaved and activated. In some cells, these initiator caspases are sufficient to cleave and activate the terminal executioner molecules, caspase-3, caspase-6, and caspase-7. However, some cells also require activation of the mitochondria-based apoptosis system to amplify the death receptor signal. Caspase-8 and caspase-10 cleave and activate the cytoplasmic proapoptotic factor Bcl-2 interacting domain (BID), which translocates to the mitochondria and induces release of the apoptosis initiating factor cytochrome c (E. K. Rowinsky, Clin. Oncol. 2005, 23: 9394-9407). A number of different receptors of the death receptor family as well as their natural ligands are known (Table 1.):
TABLE 1Death receptors and their ligandsReceptorLigandTNFR1 (DR1)TNF-α, LT-αFAS (CD95, APO-1, DR2)FasLTRAIL-R1 (DR4)TRAILTRAIL-R2 (DR5)TRAILDR3 (APO2)TL1, VEGIDR6?NGFRNGFEDAREda
While many cytotoxic agents like cis-platinum or doxorubicin as well as radiation therapy appear to activate the intrinsic apoptosis pathway, targeted induction of apoptosis via the extrinsic apoptotic pathway represents an unexploited but emerging therapeutic strategy to destroy cancer cells. The activation of cell surface receptors by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) results in direct stimulation of apoptotic signaling pathways (extrinsic stimulation). Molecules that directly activate these receptors, such as agonistic monoclonal antibodies to the TRAIL or Fas receptor (such as CH-11) and recombinant TRAIL or Fas Ligand (FasL), are being evaluated as potential monotherapies and as part of combination therapies with existing chemotherapeutic drugs and other therapeutic modalities.
TRAIL, TNFβ, FasL, and other TNF superfamily ligands demonstrated the ability to both initiate apoptosis and kill transformed cells, virally infected cells, and chronically activated T cells and B cells (S. R. Wiley et al., Immunity 1995, 3: 673-682; P. T. Daniel and P. H. Krammer, J. Immunol. 1994, 152: 5624-5632).
However, systemic therapeutic administration of recombinant TNFα (and similarly TRAIL in animal studies) in cancer patients results in a massive inflammatory response, as well as direct hepatotoxicity (A. L. Jones and P. Selby, Cancer. Surv. 1989, 8: 817-836).
One version of recombinant TRAIL, Apo2L/TRAIL (PRO1762), is currently being studied in phase I clinical trials. (A. Almasan and A. Ashkenazi, Cytokine Growth Factor Rev. 2003, 14: 337-348).
HGS-ETR1 (mapatumumab; Human Genome Sciences), a fully human agonistic monoclonal antibody that targets TRAIL-R1, is in phase II evaluations in patients with advanced malignancies. Fully human monoclonal antibodies to TRAIL-R2 (HGS-ETR2; Human Genome Sciences), such as HGS-TR2J, have also entered the clinic and are currently in phase I clinical development. HGS-ETR2 and HGS-TR2J have slightly different physiochemical and kinetic profiles that warrant the exploration of both in the clinic (R. Humphreys, et al., Ann. Oncol. 2004, 15: iii102, abstr. 383PD; R. Humphrey, et al., Presented at the 16th EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics, Geneva, Switzerland, Sep. 28-Oct. 1, 2004, abstr 204).
Especially promising appear therapeutic strategies where agents are combined that activate both the extrinsic and intrinsic pathway. Thus, ETR1 or ETR2 display synergies with cis-platin, camptothecin, topotecan, doxorubicin, gemicitabine, FU, vincristine, or paclitaxel in vitro, showing even activity in cell lines that do not go into apoptosis by treatment with either of the single agents (E. K. Rowinsky, Clin. Oncol. 2005, 23: 9394-9407).
Therefore, it appears highly desirable to find small molecules that are agonists of the FAS receptor and this receptor and its agonistic anti-body CH-11 shall provide an example for validating the method to find small molecule receptor ligand mimetics according to the present invention.
Multi-drug resistance (MDR). MDR is a major obstacle to the effective treatment of cancer. Drug resistant cancers are either inherently untreatable (intrinsic resistance) or they have progressed to develop resistance to a wide variety of anticancer agents over the course of treatment (acquired resistance). The term MDR is used to describe the ability of tumor cells exposed to a single cytotoxic agent to develop resistance to a broad range of structurally and functionally unrelated drugs. Numerous mechanisms are known to contribute to this phenomenon, including overexpression of drug efflux pumps, increased activity of DNA repair mechanisms, altered drug target enzymes, and overexpression of enzymes involved in drug detoxification and elimination. Because most chemotherapy approaches ultimately elicit their effects via apoptosis, alterations at the level of apoptosis control provide yet another mechanism by which drug resistance may occur. This review will focus on some of the strategies that have been used in an attempt to chemosensitize resistant tumors by manipulating dysregulated apoptosis pathways.
Mechanisms of drug resistance may be mediated by alterations in apoptosis pathways: the decision as to whether a cell undergoes apoptosis or continues to progress through the cell cycle is dependent on the interplay of a complex set of genes and proteins that interact to regulate cell cycle progression. Drug resistance can emerge if cells alter the expression of proteins that regulate the propagation of signals arising from cellular insults, such as chemotherapy, to protect against apoptosis. Although many details of the apoptotic pathway are still not completely understood, several proteins are known to be important regulators of this process.
A small molecule prototype. To validate the method to find small molecule receptor ligand mimetics according to the present invention and to demonstrate its utility, we further selected a small molecule well established in the art. This molecule is referred to herein as “AP-121” (alternative designations are edelfosine, ET-18-OCH3, 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine, rac-1-O-octaclecyl-2-O-methyl-glycero-3-phosphocholine or some times alkyllysophospholid or ALP). AP-121 is an etherlipid, more precisely an alkyl-lysophospholipid, having the following chemical formula:

AP-121 is a synthetic analog of platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), which is generally believed to be involved in a variety of physiological processes, such as inflammation, the immune response, allergic reactions, reproduction and also have been shown to be effective as anti-tumor agent in mammals.
Cancer chemotherapy generally aims to slow the growth of, or destroy, cancer cells while avoiding collateral damage to surrounding cells and tissues. Consequently, the most effective anticancer agents are those that are able to selectively target cancer cells while leaving normal cells relatively unaffected.
Etherlipids are known to be effective anticancer agents in vitro and in animal models. Several mechanisms of action have been proposed for the toxicity of etherlipids towards cancer cells, including the cells' lack of alkyl cleavage enzymes. The resultant inability to metabolize the etherlipids leads to their intracellular accumulation and to consequent damage to cell membrane lipid organization. Other potential mechanisms of etherlipid action include effects on levels of intracellular protein phosphorylation, and disruption of cellular lipid metabolism. Normal cells typically possess the means to avoid or overcome the potentially toxic effects of etherlipids, while cancer cells do not.
AP-121 appears to exhibit multiple biological activities, including inhibition of the PI3K-Akt/PKB survival pathway, interaction with protein kinase C, intracellular activation of the Fas/CD95 death receptor, intracellular acidification, promotion of cytokine production, and altered plasma membrane function and lipid synthesis (G. Arthur and R. Bittman, Biochim. Biophys. Acta 1998, 1390: 85-102; ID, Berkovic, Gen. Pharmacol. 1998, 31: 511-517).
A liposomal formulation of AP-121, ELL-12 was found to be more effective than free AP-121 against leukemia, lung cancer metastases, and melanoma at lower and nontoxic dosing schedules (I. Ahmad et al., Cancer Res. 1997, 57: 1915-1921).
In a further example, AP-121 has been administered per orally dissolved in milk to patients with advanced non-small cell lung cancer in a phase II trial (P. Drings et al., Onkologie 1992, 15:
In particular, the in vitro and some of the animal data is suggesting a very broad “unspecific” activity in a range of tumors—which clearly could not be proven in the published human trials. In addition, some of the biological data obtained is also contradictory which may be attributed to the different assay systems, such as different cell lines that have been used, or the different experimental conditions or the skills of the involved scientists.
For example, AP-121 has been described and is sold as an experimental molecule that is an activator of membrane bound protein kinase C (PKC) in HL-60 cells at a concentration of 20 μg/ml (E. C. Heesbeen et al., FEBS Lett. 1991, 290: 231-4). AP-121 has been found to be competitive with phosphatidylserine binding to the regulatory domain of PKC. AP-121 was also described as an inhibitor of PKC (L. W. Daniel et al., Cancer Res. 1995, 55: 4844-9). By others it was found that PKC is inhibited at medium AP-121 concentrations, but activated at low and high concentrations (J. D. Aroca et al., Eur. J. Biochem. 2001, 268: 6369-78). Others describe that PKC is not involved in the cytotoxic action of AP-121 in HL-60 and K562 cells (E. C. Heesbeen et al., Biochem. Pharmacol. 1994, 47: 1481-8). AP-121 progressively inhibited the activity of PKC alpha as the concentration was increased up to 30 mol % of the total, lipid, above which the effect was one of activation. On PKC epsilon AP-121 had a triphasic effect, activating the enzyme at low concentrations, inhibiting it at slightly higher concentrations and then activating it again at higher concentrations (P. Conesa-Zamora et al., Biochim. Biophys. Acta 2005, 1687: 110-9).
AP-121 has been also described to inhibit the association between Ras and Raf-1, a know protein interaction that promotes tumor growth (P. Samadder et al., Anticancer Research 2003, 23: 2291-5). Sub-micromolar doses of AP-121 induced rapid internalization, but not activation, of epidermal growth factor receptor (EGFR), a known anti-cancer target, and concomitant MAPK/ERK activation in A431 cells (G. A. Ruiter et al., int. J. Cancer 2002, 102: 343-50). AP-121 reduced the number of receptor sites without affecting the affinity of EGF receptors in breast cancer MCF-7 and ZR-75-1 cell lines. When added at micromolar concentrations (5-25 μM), AP-121 inhibits growth factor-induced MAPK/ERK activation, at nM doses (10-500 nM). The activation of the MAPK/ERK pathway by AP-121 in A431 cells without stimulating cell proliferation: strikingly, AP-121 (500 nM) also triggered rapid clustering and internalization, of the EGFR in A431 cells (H. Kosano and O. Takatani, Cancer Research 1988, 48: 6033-6).
AP-121 has been also described to inhibit the phosphorylation and activation of p70 S6 kinase in MCF-7 cells (G. Arthur et al., Anticancer Research 2005, 25: 95-100). Inhibition of Na,K-ATPase and sodium pump by AP-121 was described by others (K. Oishi of al., Biochem. Biophys. Res. Commun. 1988, 157: 1000-6). Activation c-Jun NH2-terminal kinase and subsequent stimulation of apoptosis by A-121 was described (C. Gajate et al., Mol. Pharmacol. 1998, 53: 602-12).
Inhibition of PI3K-AKT/PKB pathway by AP-121 seems important, as increased activity of Akt and PI3K and mutations in PTEN, its negative regulator, are associated with malignancy and render cells insensitive to apoptosis induction (M. I. Berggren, et al., Cancer Research 1993, 53: 4297-302). Thus, AP-121 may inhibit functional PI3K activation by insulin. Downstream of AKT, AKT deactivates SEK-1, which is an activator of SAPK/JNK cascade. Thus, deactivating AKT with AP-121 allows activating the pro-apoptotic SAPK/JNK proteins (see Table 2, G. A. Ruiter, et al., Anticancer Drugs 2003, 14: 167-73).
TABLE 2ED50 values (μM) for induction of apoptosis by ALP's AP-121, HePC and perifosine in the human epithelial carcinoma cell linesA431 and HeLa (mean ± SD from three independent experiments.AP-121HePCPerifosineA43115.47 ± 2.917.27 ± 3.023.17 ± 2.7HeLa 5.17 ± 1.6 8.17 ± 0.4 9.27 ± 1.8
Phospholipase C inhibition by AP-121 was described, leading to a Muscarinic receptor 1 and opiod receptor δ block, with a potential application of AP-121 in antinociception. (L. F. Horowitz et at, J. Gen. Physiol 2005, 126: 243-62).
Apoptosis triggered by AP-121 is prevented by increased expression of Phosphocholine Cytidyltransferase (CTP) (I. Baburina and S. Jackowski, J. Biol. Chem. 1998, 279: 2169-2173), suggesting CTP as a primary target of AP-121.
A connection was made to the inhibition of sphingosine-1-phosphate (S1P) S1P1 receptor mediating suppression of T cell migration (G. Dorsam et al., J. Immunol. 2003, 171: 3500-7). S1P prevents the hallmarks of apoptosis resulting from elevated levels of ceramide induced by TNFα, anti-Fas antibody, sphingomyelinase, or cell-permeable ceramide. Up-regulation of the sphingosine kinase enzyme responsible for its production may contribute to MDR by protecting the cell from apoptosis (O. Cuvillier et al., Nature 1996, 381: 800-3). Jurkat cells treated with anti-Fas monoclonal antibody (clone CH-11) underwent extensive cell death within 3 h. Fas ligation induces SAPK/JNK activation in Jurkat T cells. Fas ligation induces PARP cleavage, through a significant increase in caspase-3, caspase-6 and caspase-7 proteolytic PARP activity—S1P markedly decreased caspase-3, caspase-6 and caspase-7 activity (O. Cuvillier et al., J. Biol. Chem., 1998, 273: 2910-16).
Gajate published on the ability of AP-121 to induce clustering of the FAS receptor in lipid rafts reasoning that this results in the ability of AP-121 to induce apoptosis (C. Gajate et al., J. Exp. Med. 2004, 200: 353-365). This putative AP-121 target was also shown in lymphoma cells and cell lines (C. Gajate and F. Mollinedo, Blood 2001, 98: 3860-3863). On the other hand, Spiegel and coworkers attribute these pro-apoptotic properties to the release of cytochrome c from mitochondia (O. Cuvillier et al., Blood 1999, 94: 3583-3582), independent of the FAS receptor. AP-121 is internalized into lipid rafts of tumor cells via endocytosis (A. H. van der Luit et al., J. Biol. Chem. 2002, 277: 39541; A. H. van der Luit, et al., J. Biochem. 2003, 374: 747). Once in the cell, AP-121 triggers recruitment of Fas-associated death domain protein, procaspase-8 and procaspase-10, c-Jun N-terminal kinase, and Bid, the molecules critical for initiation of apoptosis (C. Gajate et al., Exp. Med. 2004, 200: 353). Thus, death receptor (FAS-R) and mitochondrial apoptotic routes are spaciously linked, resulting in disruption of the mitochondrial transmembrane potential, production of reactive oxygen species, caspase-3 activation, cleavage of poly(ADP-ribose) polymerase, and DNA fragmentation (C. Gajate et al., Int. J. Cancer 2000, 86, 208.).
Compared to the reported biological activities of AP-121 in cell lines which is in the low micromolar range, the most potent effect described to date to our present knowledge is the anti-angiogenic activity of 60 nanomolar on HMEC1 endothelial cells (D. Candal, Cancer Chemother. Pharmacol. 1994, 34: 175-178).
Based on these biological data on various cells and cell lines, it appears that a method is needed to discover a biologically effect of AP-121, if there is any, that is of therapeutic relevance for a specific therapeutic indication.
Despite the uncertainty of its biological effect, U.S. Pat. No. 5,149,527 describes immunopotentiating compositions comprising etherlipids as suitable agents giving rise to tumor necrosis and/or regression in subjects who have previously received successful therapy, which destroys tumors and stimulates cytotoxic macrophages. The agents should be administered at a time when formation of macrophages specifically cytotoxic for the tumor have been generated by previous therapy. However, no data has been provided to support this activity of AP-121.
AP-121 has been described as a compound useful to treat cancer (DE 02 619 686) by administering to the mammals a pharmaceutically effective amount of AP-121 to reduce the size of tumors has been also described in U.S. Pat. No. 6,514,519 and EP 1 079 838, being especially suitable for the treatment of primary and secondary malignant brain tumors.
U.S. Pat. No. 6,235,729 is directed to a method of inhibiting tumor progression in slowing or inhibiting tumor invasiveness and metastasis, comprising the step of administering to said individual a pharmacologically effective dose of a phospholipase C inhibitor, such as AP-121. Preferably, the phospholipase C inhibitor decreases phospholipase Cy. Generally, such a phospholipase C inhibitor would be administered in a dose of from about 0.1 mg/kg to about 2 mg/kg. While there is no data provided to support the claim, the dose reported will not reflect the low activity of AP-121, found in in vitro tests elsewhere.
AP-121 has been also reported to be useful to treat multiple sclerosis (MS; EP 2 363 901, DE 03 530 767) or rheumatoid arthritis (RA) or ankylosing spondylitis (AS; EP 474 712), by inhibition of activated CD4+ or CD8+ cytotoxic T-lymphocytes (CTL).